Replication-competent herpes simplex virus mediates destruction of neoplastic cells

ABSTRACT

A method for killing malignant brain tumor cells in vivo entails providing replication competent herpes simplex virus vectors to tumor cells. A replication competent herpes simplex virus vector, with defective expression of the γ34.5 gene and the ribonucleotide reductase gene, specifically destroys tumor cells, is hypersensitive to anti-viral agents, and yet is not neurovirulent.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to the use of an altered herpessimplex virus that is capable of killing tumor cells. More specifically,the present invention relates to a mutated, replication-competent HerpesSimplex Virus-1 (HSV-1) which contains mutations in two genes, ishypersensitive to antiviral agents such as acyclovir, is notneurovirulent and does not replicate in non-dividing cells, yet can killnervous system tumor cells.

[0002] Malignant tumors of the nervous system usually are fatal, despitemany recent advances in neurosurgical techniques, chemotherapy andradiotherapy. In particular, there is no standard therapeutic modalitythat has substantially changed the prognosis for patients diagnosed withmalignant brain tumors. For example, high mortality rates persist inmalignant medulloblastomas, malignant meningiomas andneurofibrosarcomas, as well as in malignant gliomas.

[0003] Gliomas are the most common primary tumors arising in the humanbrain. The most malignant glioma, the glioblastoma, represents 29% ofall primary brain tumors, some 5,000 new cases per year in the UnitedStates alone. Glioblastomas are almost always fatal, with a mediansurvival of less than a year and a 5-year survival of 5.5% or less.Mahaley et al., J. Neurosurg. 71: 826 (1989); Shapiro, et al., J.Neurosurg. 71: 1 (1989); Kim et al., J. Neurosurg. 74: 27 (1991). Afterglioblastomas are treated with radiotherapy, recurrent disease usuallyoccurs locally; systemic metastases are rare. Hochberg et al., Neurology30: 907 (1980). Neurologic dysfunction and death in an individual withglioblastoma is due to the local growth of the tumor.

[0004] In the past, viruses have been tested for their ability to treatvarious types of tumors in animals or humans. The proposed therapeuticmechanisms of viral cancer therapy in the prior art includes: (i)producing new antigens on the tumor cell surface to induce immunologicrejection, a phenomenon called “xenogenization”, and (ii) direct cellkilling by the virus, called oncolysis. Austin et al., Adv. Cancer Res.30: 301 (1979); Kobayashi et al., Adv. Cancer Res. 30: 279 (1979);Moore, Progr. Exp. Tumor Res. 1:411 (1960). Treatments for tumors inboth animals and in humans have been based on wild-type virus, passageattenuated virus, or infected cell preparations. Kobayashi, Adv. CancerRes. 30: 279 (1979); Cassel et al., Cancer 52: 856 (1983); Moore, Prog.Exp. Tumor Res. 1: 411 (1960).

[0005] Several animal models and animal tumors have been used to studyoncolysis with wild-type viruses. Moore, Ann. Rev. Microbiol. 8: 393(1954); Moore, Progr. Exp. Tumor Res. 1:411 (1960). At least nineviruses have been shown to be capable of inducing some degree of tumorregression in a variety of tumors in mice, rats, rabbits, and guineapigs. A major drawback seen in these early animal studies, however, wassystemic infection by the virus.

[0006] To avoid systemic infection, the genetic engineering of virusesfor use as antineoplastic agents has focused on generating alteredviruses that are not capable of replication in non-dividing cells.Viruses capable of replication in dividing cells preferentially infectrapidly dividing tumor cells because they are incapable of replicatingin non-dividing normal cells.

[0007] The use of replication-incompetent or defective viruses, whichrequire helper virus to be able to integrate and/or replicate in a hostcell, was hoped to prevent damage to non-tumor cells. Thereplication-defective herpes simplex virus vector system consists of anamplicon plasmid which, in herpes simplex virus infected cells, isreplicated and packaged into viral particles. Defective herpes simplexvirus vectors require helper virus to generate a herpes simplex virusvector.

[0008] The use of replication-defective retroviruses for treatingnervous system tumors requires producer cells and has been shown to belimited because each replication-defective retrovirus particle can enteronly a single cell and cannot productively infect others thereafter.Because these replication-defective retroviruses cannot spread to othertumor cells, they would be unable to completely penetrate a deep,multilayered tumor in vivo. Markert et al., Neurosurg. 77: 590 (1992).

[0009] Clinical trials employing retroviral vector therapy treatment ofcancer have been approved in the United States. Culver, Clin. Chem 40:510 (1994). Retroviral vector-containing cells have been implanted intobrain tumors growing in human patients. Oldfield et al., Hum. Gene Ther.4: 39 (1993). These retroviral vectors carried the HSV-1 thymidinekinase (HS-tk) gene into the surrounding brain tumor cells, whichconferred sensitivity of the tumor cells to the anti-herpes drugganciclovir. Of eight patients with recurrent glioblastoma multiforme ormetastatic tumors treated by stereotactic implantation of murinefibroblast cells producing retroviral vectors, five patientsdemonstrated some evidence of anti-tumor efficacy but none were cured.Culver, supra (1994). Some of the limitations of current retroviralbased therapy as described by Oldfield are (1) the low titer of virusproduced, (2) virus spread limited to the region surrounding theproducer cell implant, (3) possible immune response to the producer cellline, (4) possible insertional mutagenesis and transformation ofretroviral infected cells, (5) single treatment regimen of pro-drug,ganciclovir, because the “suicide” product kills retrovirally infectedcells and producer cells and (6) the bystander effect limited to cellsin direct contact with retrovirally transformed cells. Bi, W. L. et al.,Human Gene Therapy 4:725 (1993).

[0010] In the early 1990's, the use of genetically engineeredreplication-competent HSV-1 viral vectors was first explored in thecontext of antitumor therapy. Martuza et al., Science 252: 854 (1991). Areplication-competent virus has the advantage of being able to enter onetumor cell, make multiple copies, lyse the cell and spread to additionaltumor cells. A thymidine kinase-deficient (TK) mutant, dlsptk, was ableto destroy human malignant glioma cells in an animal brain tumor model.Martuza, supra (1991). Unfortunately, the dlsptk mutants were onlymoderately attenuated for neurovirulence and produce encephalitis at thedoses required to kill the tumor cells adequately. Markert et al.,Neurosurgery 32: 597 (1993). Residual neurovirulence, as evidenced by a50% lethality of intracranially-administered, replication-deficientherpes simplex virus viral vectors at 10⁶ plaque forming units (pfu)limits the use of such vectors for tumor therapy. Furthermore, known TKHSV-1 mutants are insensitive to acyclovir and ganciclovir, the mostcommonly used and efficacious anti-herpetic agents.

[0011] Therefore, it remains of utmost importance to develop a safe andeffective viral vector for killing tumor cells. Even though variousattempts have been made to engineer a viral vector able to kill humantumor cells in vivo, no viral vector has provided attenuatedneurovirulence at the dose required to kill tumor cells while exhibitinghypersensitivity to antiviral agents and an inability to revert towild-type virus currently, no viral vector has been demonstrated to meetthese criteria.

SUMMARY OF THE INVENTION

[0012] It is therefore an object of this invention to provide areplication-competent viral vector, suitable for use in humans, that iscapable of killing human tumor cells in vivo, that exhibitshypersensitivity to anti-viral agents and an inability to revert towild-type virus, and that is not neurovirulent at a dose required tokill tumor cells.

[0013] It is another object of the present invention to provide for theproduction of a replication-competent, herpes simplex virus-derivedvector that is effective and safe for use in the treatment of malignantbrain tumors in humans.

[0014] It is a further object of the invention to provide a safe,mutated HSV-1 vector, for use in the context of a vaccine or tumortherapy, which vector is incapable of reverting to wild-type formthrough a spontaneous single mutation.

[0015] Still another object of the present invention is to provide amutant HSV-1 vector that can selectively replicate in and kill a tumorcell of non-nervous tissue origin.

[0016] An additional object of the present invention is the productionof a replication-competent viral vector, derived from herpes simplexvirus, which can be employed in a genetic therapy against tumors byexpressing foreign genes to target an immune response that kills thetumor cells.

[0017] Yet another object of the present invention is the production ofa mutant herpes simplex virus vector containing a tumor cell-specificpromoter so that the vector can be targeted to specific tumor cells.

[0018] It is also an object of the present invention to provide forproduction of a replication competent viral vector that is effective andsafe for use as a vaccine to protect against infection by herpes simplexvirus.

[0019] In satisfying these and other objects, there has been provided,in accordance with one aspect of the present invention, areplication-competent herpes simplex virus that is incapable ofexpressing both (i) a functional γ34.5 gene product and (ii) aribonucleotide reductase. In a preferred embodiment, the vector containsalterations in both genes.

[0020] In accordance with another aspect of the present invention, amethod has been provided for killing tumor cells in a subject,comprising the step of administering to the subject a pharmaceuticalcomposition comprising (A) a herpes simplex virus vector that is alteredin (i) the γ34.5 gene, and (ii) the ribonucleotide reductase gene; and(B) a pharmaceutically acceptable vehicle for the vector, such that thetumor cells are altered in situ by the vector and the tumor cells arekilled. The tumor cells can be of a nervous-system type selected fromthe group consisting of astrocytoma, oligodendroglioma, meningioma,neurofibroma, glioblastoma, ependymoma, Schwannoma, neurofibrosarcoma,and medulloblastoma. Other kinds of tumor cells which can be killed,pursuant to the present invention, include those selected from the groupconsisting of melanoma cells, pancreatic cancer cells, prostatecarcinoma cells, breast cancer cells, lung cancer cells, colon cancercells, lymphoma cells, hepatoma cells and mesothelioma and epidermoidcarcinoma cells.

[0021] In accordance with still another aspect of the present invention,a method is provided for killing tumor cells in a subject, comprisingthe steps of administering to the subject a herpes simplex virus vector,wherein the vector comprises a tumor cell-specific promoter wherein thepromoter controls expression of at least one viral protein necessary forviral replication and wherein the promoter is induced selectively or ata higher level in tumor cells than in normal cells. This method canential the use of a promoter that is selectively capable of expressionin nervous-system tumor cells, for example, glioblastoma cells,medulloblastoma cells, meningioma cells, neurofibrosarcoma cells,astrocytoma cells, oligodendroglioma cells, neurofibroma cells,ependymoma cells and Schwannoma cells.

[0022] A method also in provided for preparing a replication-competentvector of a herpes simplex virus, comprising the steps of (A) isolatinga viral genome of the herpes simplex virus; and (B) permanently alteringthe genome so that the virus is (1) sensitive to antiviral agents, (2)kills tumor cells and (3) expresses decreased generalizedneurovirulence. For example, the the herpes simplex virus of the vectorcan be HSV-1 or HSV-2.

[0023] The present invention further provides for a method of protectinga subject against herpes simplex virus infection, comprising the step ofadministering to the subject a pharmaceutical composition that iscomprised of (A) a herpes simplex virus vector wherein the genome of thevirus is altered in (i) the γ34.5 gene, and (ii) the ribonucleotidereductase gene; and (B) a pharmaceutically acceptable vehicle for thevector.

[0024] According to still another aspect of the present invention, therehas been provided a method of eliciting an immune response to a tumorcell, comprising the step of administering to the subject apharmaceutical composition comprising (A) a herpes simplex virus,wherein the genome of the virus (i) contains an expressible non-herpessimplex virus nucleotide sequence encoding a desired protein capable ofeliciting an immune response in the subject, and (ii) is altered in theγ34.5 gene, and the ribonucleotide reductase gene; and (B) apharmaceutically acceptable vehicle for the virus. In a preferredembodiment, the method further comprises the step of co-administrationwith neurosurgery, chemotherapy or radiotherapy.

[0025] A mutant viral vector of the present invention is sensitive totemperatures greater than the basal temperature of the host, whichprovides an additional safety feature by further compromising viralreplication in the presence of encephalitis and fever.

[0026] Other objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention can be understood more fully by referenceto the following drawings, where:

[0028]FIG. 1 is a schematic illustration of the construction of a mutantherpes simplex virus containing a 1 kB deletion in both copies of theγ34.5 gene and an insertion in the ICP6 gene.

[0029]FIG. 2 shows the sequence arrangement of a mutant herpes simplexvirus, G207-1, compared to its parental wild-type background (strain F).The abbreviations are B, BamHI; Be, BstEII; G, BglIII; N, NcoI; S, ScaI;St, StuI; and X, XhoI.

[0030]FIG. 3. is a graph illustrating the ability of G207-1 and G207-2to kill all human U-87MG glioma cells in culture, including at lowmultiplicity of infection (MOI=0.01).

[0031]FIG. 4 is a graph illustrating the ganciclovir (GCV) sensitivityof R3616, G207-1, and G207-2 which reveals that G207-1 and G207-2 areten-times more sensitive to ganciclovir than R3616. R3616 (strain F) hasthe same sensitivity to ganciclovir as strain KOS (wild-type).

[0032]FIG. 5 is a graph illustrating the ability of G207-1 and G207-2 toinhibit the growth of human brain tumor cells (U-87MG) in thesubcutaneous human brain tumor model in athymic mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The present invention exploits the ability of mutant,replication-competent HSV-1 to enter a tumor cell in situ, make multiplecopies, lyse the tumor cell and spread to additional tumor cells withrelatively minor effects on the surrounding normal cells. The mutantherpes simplex virus of the present invention has each of the followingcharacteristics: (1) efficacy in killing human brain tumor cells, (2)marked attenuation of generalized neurovirulence to protect the normalbrain, (3) multiple deletions so that a single mutation cannot causereversion to the wild-type viral phenotype, and (4) hypersensitivity toganciclovir so that undesired spread of the virus can be prevented. Themutant virus of the present invention is capable of replicating inneoplastic cells but spares surrounding non-neoplastic tissue.

[0034] Viruses of the instant invention are engineered to containalterations in the expression of at least two specific HSV-1 genes: (1)the γ34.5 gene and (2) the ribonucleotide reductase gene. Alterations inthis regard include any that disrupt the expression of the product ofboth of the γ34.5 gene and the ribonucleotide reductase gene. Thepresence of such multiple mutations further reduces the possibility ofreversion to wild-type pathogenicity. The present invention providesmethods for sequentially constructing and testing viruses for theability to effectively kill brain tumor cells without harmingsurrounding normal brain. Additionally, mutations can be inserted intothese vectors to increase their sensitivity to systemically administereddrugs.

[0035] Herpes Simplex Virus Vectors with Single Alterations in theRibonucleotide Reductase or γ34.5 Gene

[0036] Initial work on the use of attenuated herpes simplex virusvectors for use in anti-tumor therapy employed HSV-1 mutated in one geneallowing the vector to replicate in dividing cells, but not innon-dividing cells. Two such single gene-mutant herpes simplex virusvectors are (1) hrR3, deficient in ribonucleotide reductase, containingan Escherichia coli lacZ gene insertion in the ICP6 gene that encodesthe large subunit of RR, [Mineta, T. et al., Gene Therapy 1:S78 (1994)and Mineta et al., J. Neurosurg. 80: 381 (1994)]; and (2) R3616, whichcontains mutations in both copies of the γ-34.5 gene. Markert et al.,Neurosurgery 32: 597 (1993).

[0037] Mutants of ribonucleotide reductase have been constructed by anumber of methods. The hrR3 mutant contains an Escherichia coli lacZgene insertion in the ICP6 gene, which encodes the large subunit ofribonucleotide reductase. Other ribonucleotide reductase herpes simplexvirus mutants are suitable for constructing the mutant viral vector ofthe invention. Goldstein and Weller, supra; Goldstein and Weller, supra;Preston et al., Virol. 167: 458 (1988).

[0038] Ribonucleotide reductase (RR) is a key enzyme in the de novosynthesis of DNA precursors, catalyzing the reduction of ribonucleotidesto deoxyribonucleotides. HSV-1 encodes its own RR (UL39 and UL40 genes),which is composed of two non-identical subunits. Duita, J. Gen. Virol.64: 513 (1983). The large subunit (140 k molecular weight), designatedICP6, is tightly associated with the small subunit (38 k molecularweight). Herpes simplex virus RR is required for efficient viral growthin non-dividing cells but not in many dividing cells. Goldstein andWeller, J. Virol. 62:196 (1988); Goldstein and Weller, Virol. 166: 41(1988); Jacobson et al., Virol. 173: 276 (1989). Both RR subunits arepresent in HSV-2. It is noted that HSV-1 ICP6 is the same as HSV-2ICP10. Nikas et al., Proteins 1:376 (1986); McLaughlan and Clements EMBOJ. 2: 1953 (1983); Swain and Halloway J Virol. 57: 802 (1986)3 andmutations in the small subunit of RR also leads to loss of RR activityand neuropathogenicity (Cameron et al., J. Gen. Virol. 69: 2607 (1988)].The presence of the lacZ gene in hrR3 allows identification ofvirally-infected tumor cells using β-galactosidase histochemistry.

[0039] The cytopathic effect of hrR3 (0.1 pfu/cell) on the U-87MG humanglioblastoma cell line in vitro was significant; only 0.2% of U-87MGcells were alive 67 hours post-infection. For in vivo studies, tenanimals harboring U-87MG tumors were randomly divided and treatedintraneoplastically with either 5×10⁵ plaque-forming units of hrR3 orwith medium alone. The viral treatment group showed significantinhibition of tumor growth (p<0.01, one-sided Wilcoxon rank test).

[0040] An important difference between ribonucleotide reductasedeficient (RR⁻) and other herpes simplex virus mutants is hrR3'shypersensitivity to acyclovir and ganciclovir. Because TK⁻ HSV-1 mutantsknown in the art are resistant to these anti-viral agents, such mutantscould be difficult to eliminate in the event of systemic infection orencephalitis. Thus, in the event of viral encephalitis, hrR3 isresponsive to antiviral therapy.

[0041] Also, herpes simplex virus RR- mutants are severely compromisedin their ability to produce infections and synthesize viral DNA at 39.5°C. in vitro. Goldstein and Weller, Virology 166: 41 (1988). Therefore,these mutants are attenuated for neurovirulence and less likely topropagate in the event of a fever in the infected host. Suchcharacteristics are essential to a therapeutic vector which must be ofattenuated neurovirulence and amenable to antiviral therapy in the eventof viral encephalitis.

[0042] Herpes simplex virus mutants deficient in only the γ34.5 gene,such as R3616, are attenuated for neurovirulehce, which reduces thepossible damage to normal brain cells. Goodman et al., J. Virol. 63:1153 (1989); Chou et al., Science 250: 1262 (1990). The decreasedneurovirulence of R3616 is putatively associated with the cessation ofneuronal protein synthesis, which is preempted in wild-type herpessimplex virus infection. Chou and Roizman, Proc. Nat'l Acad. Sci. USA89: 3266 (1992). The γ34.5 gene product can be detected by Western blotor ELISA analysis of infected cell proteins with antibodies or lack ofreplication in confluent primary cells. See Bolovan et al., J. Virol.68: 48 (1994). The γ34.5 gene is also present in HSV-2. McGeoch et al.,J. Gen. Virol. 72:3057 (1991). The γ34.5 gene has been sequenced in fourstrains of HSV-1, namely F, 17, MGH-10 and CVG-2. Chou and Roizman, J.Virol. 64: 1014 (1990). The γ34.5 gene mutant HSV-1 vectors retain awild-type level of sensitivity to acyclovir. Markert et al., supra(1993).

[0043] Mutants of γ34.5 have been constructed by various investigatorsusing different techniques and in different strains such as mutant 1771(McKie et al., J. Gen. Virol. 75:733 (1994)] and 17termA [Bolovan etal., J. Virol. 68: 48 (1994)] in HSV-1 strain 17.

[0044] Construction of Herpes Simplex Virus Vectors

[0045] HSV-1 is a human neurotropic virus that is capable of infectingvirtually all vertebrate cells. Natural infections follow either alytic, replicative cycle or establish latency, usually in peripheralganglia, where the DNA is maintained indefinitely in an episomal state.

[0046] Replication-competent recombinant herpes simplex virus vectors ofthe instant invention contain alterations in expression of two specificherpes simplex virus genes: (1) the γ34.5 gene and (2) theribonucleotide reductase gene. Such alterations render the product ofboth genes non-functional or reduce their expression such that themutant herpes simplex virus vector has the properties of the instantinvention. Ways to achieve such alterations include (a) any method todisrupt the expression of the product of both of these genes or (b) anymethod to render the expressed γ34.5 gene product and ribonucleotidereductase nonfunctional.

[0047] Numerous methods known to disrupt the expression of a gene areknown, including the alterations of these genes or their promotersequences in the HSV-1 genome by insertions, deletions and/or basechanges. Roizman and Jenkins, Science 229: 1208 (1985). The mutatedherpes simplex virus vector of the instant invention is a replicationcompetent herpes simplex virus whose genome is altered in the γ34.5 geneand the ribonucleotide reductase gene. Alterations in the γ34.5 gene andthe ribonucleotide reductase gene include modifications in either thestructural or regulatory sequences of these genes. Genetic alterationscan be determined by standard methods such as Southern blothybridization of restriction endonuclease digested viral DNA, sequencingof mutated regions of viral DNA, presence of reporter gene (forinsertions), new restriction endonuclease site, enzymatic assay forribonucleotide reductase activity (Huszar and Bacchetti, J. Virol.37:580 (1981)], Western blot or ELISA analysis of infected cell proteinswith antibodies to RR or γ34.5, and/or lack of replication in confluentprimary cells for γ34.5. See Bolovan et al., J. Virol. 68: 48 (1994)] ormouse cells for RR- [Jacobson et al., Virology 173: 276 (1989).

[0048] The following genetic manipulations of herpes simplex virusprovide examples to illustrate the production of mutant herpes simplexvirus vectors. The engineering of the herpes simplex virus vectors ofthe instant invention exploit two well-characterized genes, the γ34.5and ribonucleotide reductase genes, in a biologically well-characterizedvirus.

[0049] A herpes simplex virus vector that has been mutated in its γ34.5and ribonucleotide reductase genes can be isolated after mutagenesis orconstructed via recombination between the viral genome andgenetically-engineered sequences. The high rate of recombination inherpes simplex virus and the fact that transfected viral DNA isinfectious renders genetic manipulation very straightforward. Thesegenetically-altered, replication-competent viruses can be used in thesafety and efficacy assays described below.

[0050] HSV-1 contains a double-stranded, linear DNA genome, 153kilobases in length, which has been completely sequenced by McGeoch.McGeoch et al., J. Gen. Virol. 69: 1531 (1988). McGeoch et al., NucleicAcids Res 14: 1727 (1986); McGeoch et al., J. Mol. Biol. 181: 1 (1985);Perry and McGeoch, J. Gen. Virol. 69: 2831 (1988). DNA replication andvirion assembly occurs in the nucleus of infected cells. Late ininfection, concatemeric viral DNA is cleaved into genome lengthmolecules which are packaged into virions. In the CNS, herpes simplexvirus spreads transneuronally followed by intraaxonal transport to thenucleus, either retrograde or anterograde, where replication occurs.

[0051] DNA constructs employing HSV-2 based on those illustrated hereinusing the HSV-1 genome are encompassed by the present invention. HSV-2contains both RR subunits; HSV-1 ICP6 is analogous to HSV-2 ICP10. Nikaset al., Proteins 1: 376 (1986); McLaughlan and Clements, EMBO J. 2: 1953(1983); Swain and Halloway, J. Virol. 57: 802 (1986). γ34.5 is alsopresent in HSV-2. McGeoch et al., J. Gen. Virol. 72: 3057 (1991).

[0052] Impairment of Gene Expression Via Modification of γ34.5 orRibonucleotide Reductase Regulatory Sequences

[0053] Another way to render a herpes simplex virus incapable ofexpressing functional γ34.5 gene product and ribonucleotide reductase isto impair their expression. The expression of these two genes can behalted by altering the regulatory sequences of the γ34.5 andribonucleotide reductase genes.

[0054] The regulatory regions for γ34.5 and/or ribonucleotide reductasecan be altered by standard techniques to disrupt the expression of theγ34.5 and ribonucleotide reductase gene. For example, their regulatorysequences could be altered within the viral genome using techniquesdescribed above for the alteration of coding sequences.

[0055] The promoter regions of γ34.5 and ribonucleotide reductase ICP6have been mapped. The promoter for γ34.5 has been mapped to a regionwithin the “a” sequence. The “a” sequence also contains sequences forcleavage of unit length DNA from HSV-1 concatamers, packaging of HSV-1DNA into capsids and inversion of L and S components. Chou and Roizman,J. Virol. 57: 629 (1986). The promoter region of ICP6 has been mapped tothe 5′ upstream sequences of the ICP6 structural gene. Goldstein andWeller, J. Virol. 62: 196 (1988); Sze and Herman, Virus Res. 26: 141(1992). The transcription start site for the small subunit of RR, namelyUL40, falls within the coding region of ICP6. McLauchlan and Clements,J. Gen. Virol. 64: 997 (1983); McGeoch et al., J. Gen. Virol. 69: 1531(1988).

[0056] The effect of these alterations on the regulatory capacity ofγ34.5 and RR genes can be detected by inserting a reporter genedownstream of the promoter, such as that described for the ICP6/lacZfusion. Goldstein and Weller, J. Virol. 62: 196 (1988); Sze and Herman,Virus Res. 26: 141 (1992). Because herpes simplex virus genes areregulated differently when present in the cellular genome, the effectsof each alteration in the γ34.5 or ribonucleotide reductase regulatorycomponent would be assessed in various mammalian target cells. McKnightet al.,in CANCER CELLS 4; DNA TUMOR VIRUSES, Cold Spring Harbor (1986)163-173.

[0057] Additional methods for the construction of engineered viruses areknown in the art. Additional methods for the genetic manipulation of DNAsequences are known in the art. Generally, these include Ausubel et al.,chapter 16 in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley andSons, Inc.); Paoletti et al., U.S. Pat. No. 4,603,112 (July 1986).Virological considerations also are reviewed in Coen D. M., “MolecularGenetics of Animal Viruses,” in VIROLOGY 123-150 (2nd ed.) (Raven Press,1990).

[0058] The construction of HSV-1 vectors is described, for example, inU.S. Pat. No. 5,288, 641; Roizman and Jenkins, J. Science 229: 1208(1985); Johnson et al., J. Virol. 66: 2952 (1992); Gage et al., J.Virol. 66: 5509 (1992); Spaete and Frenkel, Cell 30; 295 (1982);Goldstein and Weller, J. Virol. 62: 196 (1988), Coen, chapter 7,Virology, Raven Press, 1990; Breakefield and DeLuca, The New Biologist,3:203 (1991); Leib and Olivo, BioEssays 15:547 (1993); Glorioso et al.,Seminars in Virology 3:265 (1992); Chou and Roizman, Proc. Natl. Acad.Sci. USA, 89:3266 (1992); Breakfield et al., Molec. Neurobiol. 1: 339(1987); Shih et al., in: VACCINES 85, Cold Spring Harbor Press (1985)177-180; Palella et al., Molec. Cell. Biol. 8: 457 (1988); Matz et al.,J. Gen. Virol. 64: 2261 (1983); Mocarski et al., Cell 22: 243 (1980);and Coen et al., Science 234: 53 (1986).

[0059] Imparting Hypersensitivity to Antiviral Agents

[0060] One safety precaution in the therapeutic use of herpes simplexvirus against gliomas involves providing a means to stop any potentialinfection of other dividing cells. Clinical studies indicate that evenwild-type HSV-1 viruses generally do not spread far from the site ofinitial infection or cause serious systemic disease in immunocompetentindividuals. Sacks et al., Ann. Int'l Med. 111: 893 (1989).

[0061] It is noted that TK viruses have sometimes been associated withprogressive disease in certain immunocompromised patients and that theHSV-1 mutant dlsptk is resistant to acyclovir. Erlich et al., New Engl.J. Med. 320: 293 (1989); Coen et al., Proc. Nat'l Acad. Sci. USA 86:4736 (1989). Any mutant replication-competent viral vector that is moresensitive to the anti-viral agent than its wild-type parent is deemedhypersensitive to the anti-viral agent, potentially providing a means toabort an undesired spread of the mutant virus.

[0062] In constructing herpes simplex virus mutants for use in vivo, themutants are tested for their sensitivity to current anti-herpetic drugtherapies in order to control unforeseen virulent infections. A numberof drugs currently are available to treat herpes infections in humans,the most effective being nucleoside analogs which block herpes simplexvirus DNA replication. Three herpes simplex virus genes are known to beinvolved in sensitivity to nucleoside analogs: herpes simplex virus DNApolymerase (UL30, pol), herpes simplex virus thymidine kinase (UL23,tk),and CNV UL97 which shares homology with protein kinases and bacterialphosphotransferases. Furman et al., J. Virol. 32: 77 (1979); Littler etal., Nature 358: 160 (1992); Sullivan et al., Nature 358: 162 (1992).

[0063] There are a number of herpes simplex virus DNA polymerase mutantswhich exhibit hypersensitivity to ganciclovir, including PAA^(r)5 andAraA^(r)9. Coen et al., J. Virol. 53: 477 (1985). Unfortunately,intracranial injections of AraA^(r)9 led to premature death and had noeffect on subcutaneous tumor growth. Markert et al., supra. Anothermutant herpes simplex virus, the dlsptk virus, is no longer drugsensitive, at least to nucleoside analog drugs, and thereforepotentially uncontrollable in vivo.

[0064] Attenuation for Neurovirulence

[0065] Attenuated or decreased generalized neurovirulence means thatlife-threatening encephalitis does not ensue after infection with thedouble mutant herpes simplex virus vector of the instant invention.Because herpes simplex virus-induced encephalitis in humans is verydifficult to treat and can be fatal, even with adequate pharmacologicmeasures, decreased generalized neurovirulence is an important featureof the instant invention. The mutant virus of the present invention iscapable of replicating in neoplastic cells but spares surroundingnon-neoplastic tissue.

[0066] Different herpes simplex virus strains vary in neurovirulence andmore attenuated strains may be employed in the construction of thedouble mutant to further decrease neurovirulence. Other HSV-1 strainsavailable from ATCC include HF (ATCC VR-260), MacIntyre (ATCC VR-539),MP (ATCC VR-735) and HSV-2 strains G (ATCC VR-734) and MS (ATCC VR-540).

[0067] Alternatively, any herpes simplex virus gene mutation leading todecreased viral replication in vivo and/or in specific cell populationsmay be used in the mutated herpes simplex virus vector of the invention.Other neurovirulence genes include: (i) dUTPase [Pyles et al., J. Virol.66:6706, (1992)], (ii) UL53 [Moyal et al., Virus Res. 26:99 (1992)],(iii) a22 [Sears et al., J. Virol. 55: 338 (1985)] and (iv) US3[Meignier et al., Virology 162:251 (1988)].

[0068] From a clinical perspective, herpes simplex virus encephalitis isthe most commonly reported viral infection of the central nervous system(CNS) in the United States, with an estimated incidence of 2.3 cases permillion population. Herpes simplex virus encephalitis is usuallylocalized to the temporal lobe and the limbic system and histologicalexamination of autopsy cases demonstrates viral antigen at these sites.A number of drugs are available to control infection, includingacyclovir 9-92-hydroxyethoxy-methyl)guanine, Zovirax®, adeninearabinoside (Vidarabine®), foscarnet (phosphonoformic acid, PFA) andganciclovir 9(1,3-dehydroxy-2-propoxy)methylguanine, DHPG, 2′NDG,Cytovene®. See Whitley et al., in Lopez et al., (eds.) IMMUNOBIOLOGY ANDPROPHYLAXIS OF HUMAN HERPESVIRUS INFECTIONS, page 243 (1990, PlenumPress, N.Y.); Whitley et al., N. Engl. J. Med. 297: 289 (1977); Oberg,Pharmacol. Ther. 19: 387 (1983); DeArmond, Transplant. Proc. 23: 171(1991).

[0069] Achieving Tumor-Specificity

[0070] Because herpes simplex virus has a very broad host range andseems capable of infecting all cell types in the CNS, herpes simplexvirus mutants of the instant invention may be targeted to specific tumortypes using tumor cell-specific promoters. The term “tumor cell-specificpromoter” indicates a promoter that is induced selectively or at ahigher level in the target tumor cell than in a normal cell. Tumorcell-specific promoters include promoters that are induced selectivelyor at a higher level in a particular cell type or a tumor cell.

[0071] The vectors of the invention also can be designed to selectivelyreplicate in and kill a tumor cell of non-nervous tissue origin. Theherpes simplex virus vector of the invention is engineered to place atleast one viral protein necessary for viral replication under thecontrol of a cell specific or tumor cell-specific promoter. The tumorcell-specific promoter is induced selectively or at higher levels intumor cells than in normal cells.

[0072] Such tumor cell-specific, HSV-1 mutants utilize promoters fromgenes that are highly expressed in the targeted tumor, such as theepidermal growth factor receptor gene promoter (EGFr) or the basicfibroblast growth factor (bFGF) gene promoter or the NESTIN or othertumor associated promoters or enhancer elements to drive expression ofan essential herpes simplex virus gene (e.g., ICP4), under circumstancesin which the wild-type essential herpes simplex virus gene would not beexpressed. Rendering the essential herpes simplex virus genenon-functional can be achieved by genetic inactivation or replacement ofpromoter with tumor cell-specific promoter.

[0073] The instant invention encompasses a host-range conditional herpessimplex virus mutant where an essential viral gene product is under thecontrol of a tumor cell-specific promoter rather than its own viralpromoter. In permissive cells, containing the proper regulatory proteinsfor this specific promoter, the essential viral gene product isexpressed and the virus is able to replicate and spread to adjacentcells until a non-permissive cell is infected. These studies areapplicable to the replication-competent herpes simplex virus of thisinvention. These constructs, however, are only replication-competent inthe correct cell types (i.e., tumor cells) and are replication-deficientin other cells (i.e., surrounding tissue).

[0074] Many tumor cell types express phenotypic markers which are turnedoff in the normal, terminally-differentiated cell. One can takeadvantage of this altered expression pattern to construct tumorcell-specific viruses. Examples of such differentially regulated genesin neural tumors include: (i) nestin, an intermediate filament proteinnormally expressed in neuroepithelial stem cells, yet not in mature CNScells, which is ubiquitously expressed in human brain tumors, mostprominently in gliomas, (ii) basic fibroblast growth factor (bFGF), adifferentiation factor and mitogen for neuroectoderm, which is highlyexpressed in human gliomas and meningiomas but not in metastatic braintumors or normal brain tissue and (iii) epidermal growth factor receptor(EGFr), a membrane-bound tyrosine-specific protein kinase that isstimulated by EGF, which is very often overexpressed, altered and thegene amplified in human high grade gliomas but rarely in normal brain.

[0075] Herpes Simplex Virus Vectors Effective for Xenogenization

[0076] The mutant herpes simplex virus vector of the instant inventioncan be employed in a genetic therapy against specific tumors byexpressing foreign genes in a tumor cell-specific fashion in order totarget an immune response that kills the tumor cells. Tepper and Mulé,Human Gene Therapy 5: 153 (1994). In addition, the instant inventionemploys the replication competent-herpes simplex virus vector havingdecreased neurovirulence as a tumor cell modulator or inducer of animmune response against the tumor cells. The mutant herpes simplex virusvector of the invention can be further altered to express cytokines inthe tumor target cell in order to elicit an immune response against thetumor cells. For example, a mutant herpes simplex virus vector caninduce viral-mediated killing of tumor cells, which then is amplified bya cytokine-enhanced immune response, a cytokine having been expressed bythe vector itself. The expression of cytokines, or other gene products,from the mutant herpes simplex virus vector would occur within hours ofinfection so that sufficient gene products would be synthesized prior tocell killing. Cell killing may -even increase the efficacy of theanti-tumor immune response. Barba et al., Proc. Nat'l Acad. Sci. USA 91:4348 (1994).

[0077] Herpes Simplex Virus Vector-Mediated Destruction of Tumor Cells

[0078] Exemplary candidates for treatment according to the presentinvention include, but are not limited to (i) non-human animalssuffering from tumors and neoplasms, (ii) humans suffering from tumorsand neoplasms, (iii) animals suffering from nervous system tumors and(iv) patients having malignant brain tumor, including astrocytoma,oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma,Schwannoma, neurofibrosarcoma, and medulloblastoma.

[0079] Preferentially, the treatment will be initiated by directintraneoplastic inoculation. For tumors in the brain, MRI, CT, or otherimaging guided stereotactic technique will be used to direct viralinoculation or virus will be inoculated at the time of craniotomy.

[0080] The pharmaceutical compositions of the present invention would beadvantageously administered in the form of injectable compositions. Atypical composition for such purpose would comprise a pharmaceuticallyacceptable vehicle. For instance, the composition could contain humanserum albumin in a phosphate buffer containing NaCl. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike. See REMINGTON'S PHARMACEUTICAL SCIENCES (15th ed.) 1405-1412 &1461-1487, Mack Publishing Co. (1975), and THE NATIONAL FORMULARY XIV(14th ed.), American Pharmaceutical Association (1975). Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oil and injectable organic esters such as ethyloleate. Aqueouscarriers include water, aqueous solutions, saline solutions, parenteralvehicles such as sodium chloride, Ringer's dextrose, etc. Intravenousvehicles include fluid and nutrient replenishers. The pH and exactconcentration of the various components the pharmaceutical compositionare adjusted according to routine skills in the art. Goodman and Gilman,THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th ed.).

[0081] Typically, the herpes simplex virus vector would be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectionmay also be prepared. The preparation also may be emulsified. The activeimmunogenic ingredient is often mixed with an excipient which ispharmaceutically-acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the vector may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH-buffering agents, adjuvants orimmunopotentiators which enhance the effectiveness of the vectorvaccine.

[0082] Additional formulations which are suitable for other modes ofadministration include oral formulations. Oral formulations include suchtypical excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate and the like. The compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10%-95% of active ingredient,preferably 25-70%.

[0083] The term “unit dose” refers to physically discrete units suitablefor use in humans, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect inassociation with the required diluent, i.e., carrier or vehicle, and aparticular treatment regimen. The quantity to be administered, bothaccording to number of treatments and amount, depends on the subject tobe treated, capacity of the subject's immune system to synthesizeantibodies, and degree of protection desired. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner and are peculiar to each individual. However, suitabledosage ranges are on the order of one to several hundred micrograms ofactive ingredient per individual. Suitable regimes for initialadministration and booster shots also vary but are typified by aninitial administration followed in one or two week intervals by one ormore subsequent injections or other administration.

EXAMPLE 1 Construction of Highly Attenuated, Double HSV Mutants

[0084] Viruses and Cell Lines

[0085] HSV-1 wild-type strain (KOS or Strain F) and HSV mutants (R3616,hrR3) were kindly provided by D. M. Coen, B. Roizman, J. Chou, and S. K.Weller. HSV-1 strain F is available as ATCC VR-733; Vero cells areavailable as ATCC CRL 1587. R3616, which is derived from HSV-1 strain F,contains a 1-kilobase-pair deletion in both copies of the γ34.5 gene.R3616 was constructed as described in Chou et al., Science 250: 1262(1990).

[0086] Stocks of viruses were generated in African green monkey kidneycell (Vero) cultures as described. Virus stocks were prepared asdescribed by Coen et al., J. Virol. 53:477 (1985).

[0087] Human glioblastoma cells U-87MG, T98G, U-138MG, and A172 wereobtained from American Type Culture Collection (Rockville, Md.) andcultured in Dulbecco's minimal essential medium (DMEM) supplemented with10% inactivated fetal calf serum (IFCS) and antibiotics.

[0088] Viral DNA is isolated from infected cells, which are gently lysedwith NP40, treated with RNase, then SDS and Proteinase K, and finallyextracted with phenol, chloroform/isoamylalcohol, and ether. The viralDNA is suitable for transfection after precipitation with ethanol andresuspension in water. For the generation of recombinant viruses, thepiece of DNA to be recombined into the viral genome is excised from aplasmid. The linear DNA is co-transfected with viral DNA into cellscapable of supporting propagation of the recombinant progeny virus. Whenextensive cytopathic effects are observed, progeny virus is harvested.Recombinant viruses are then plated on permissive cells under selectableor screenable conditions. For example, LacZ+ recombinant plaques arestained by adding X-gal and blue plaques (LacZ+) are selected. Furtherplaque purification (three times), is conducted before a stock is made.

[0089] Construction of Herpes Simplex Virus Incapable of Expressing Bothγ34.5 Gene Product and Ribonucleotide Reductase

[0090] Herpes simplex virus strains mutated in both the γ34.5 andribonucleotide reductase genes are constructed using standard proceduresfor the generation of viral recombinants as described by Goldstein andWeller. Both of these genes are non-essential for viral growth inculture and therefore null mutants are viable in culture. Such doublemutants include the insertion of the E. coli Lac Z gene in either gene,so that replication in site can readily be detected.

[0091] An exemplary mutant herpes simplex virus vector of the instantinvention can be constructed by homologous recombination using DNAisolated from R3616 virus and a 5.3 kB HindIII-XbaI fragment ofpKX2-βG3. One example of such a mutant within the present invention isdesignated “G207.” FIG. 1 illustrates the construction of G207. Fiveisolates were purified and termed G207-1, -2, -3, -4, -5.

[0092] The HSV-1 mutant R3616, derived from HSV-1 wild-type strain F,contains a 1-kB deletion in both copies of the γ34.5 gene. To constructan ICP6 lacZ insertion in R3616 viral DNA, the 5.3 kb HindIII-XbaIfragment of pKX2-βG3, which contains a lacZ insertion in the 2.3-kB XhoIfragment of ICP6 gene, was cotransfected with R3616 infectious viral DNAinto Rabbit Skin (RS) cells, and introduced into the viral DNA byhomologous recombination. Plasmid pKX2-BG3 containing a lacZ geneinsertion in the 2.3 Kb XhoI fragment of ICP6 gene (KOS), was kindlyprovided by Dr. S. K. Weller (Univ. of Connecticut). Goldstein andWeller, J. Virol. 62:196 (1988). Plasmid pKpX2′ was constructed bypartial digestion of pKX2-βG3 with BamHI, removal of lacZ gene andreligation. Plasmid pRB4081 containing NcoI-SphI fragment of γ34.5 gene,was kindly provided by B. Roizman. Chou et al., Science 25: 1262 (1990).All recombinant plasmids were propagated by standard procedures.

[0093] Two hundred to 1,000 infectious units of R3616 viral DNA(approximately 1 μg) are co-transfected with a 10-fold molar excess ofthe 5.3 kB insert of pKX2-βG3, which is excised by cutting with XbaI andHindIII, to RS cells. When wide spread cytopathic effects were observed,progeny are harvested and titers determined on Vero cells.

[0094] On day 2 or 3 following infection, plaques were stained withX-gal solution. Recombinant viruses were identified by plaques stainingpositive with X-gal. Recombinant viral plaques (γ34.5-/ICP6- and LacZ⁺)stain blue in the presence of X-gal. Recombinant virus from blue plaquesis purified and the DNA analyzed by restriction endonuclease mapping toconfirm the DNA structure of the mutant viruses. Blue plaques werepurified three times by passage in Vero cells in a limiting dilutionmethod before stocks were made.

[0095] The plaque morphology of G207-1 and G207-2 was analyzed as wellas the effect of various concentrations of IFCS-containing medium onplaque morphology. Infected vero cell monolayers were cultured at 37“C.in medium containing 0.5%, 1%, 2.5% and 5% IFCS; were fixed at 36-48 hrpost-infection; and were stained with X-gal, to detect β-gal activity,and then counterstained with neutral red.

[0096] G207-1 mutants produced non-syncytial plaques, whereas G207-2mutants produced syncytial plaques, characterized by extensive cell-cellfusion.

[0097] Table 1 documents the increasing plaque diameters underconditions of increased cell growth for G207-1 and G207-2. The diametersof plaques were measured using a micrometer under an invertedphase-contrast microscope at 40× magnification. Each value representsthe average diameter of 15 plaques. TABLE 1 Diameters of plaques invarious concentrations of IFCS medium 0.5% IFCS 1% IFCS 5% IFCS R36160.48 ± 0.13 0.44 ± 0.12 0.45 ± 0.094 (NS)  1.1 ± 0.36 (Syn) G207-1 0.42± 0.15 0.48 ± 0.16 0.63 ± 0.18 (Non-syn.) G207-2 0.45 ± 0.16 0.48 ± 0.17 1.0 ± 0.30 (Syn.)

[0098] The sequence and gene arrangement of G207 viruses compared to itsstrain F wild-type background is illustrated in FIG. 2. The boxes onFIG. 2's top line represent the inverted repeat sequence flanking thelong (UL) and short (Us) components of herpes simplex virus genome,which is represented by thin lines. The expanded domains of the longunique region show the location of the ICP6 gene. The thick line showsthe transcribed sequences of ICP6 gene. Mutant G207 contains thestructural gene of lacZ inserted into the BamHI site in the ICP6 gene.The expanded domains of the repeat regions show the location of theγ34.5 gene. Mutant G207 contains a lkB deletion in both copies of theγ34.5 gene.

[0099] Analysis of Mutant Viral DNA

[0100] In order to confirm the correctly altered structure of the herpessimplex virus vectors, Southern blot analysis was performed on themutants of invention. Viral DNAs were prepared from partially purifiedvirions. Total viral DNAs (KOS, hrR3, Strain F, R3616, and G207) weredigested with restriction endonuclease, separated by agarose gelelectrophoresis in a Tris-borate-EDTA buffer and transferred by themethod of Southern. Recombinant DNAs used as probes for hybridizationwere labeled by ECL labeling Kit (Amarsham) as suggested by thesupplier. To confirm that the viral mutants contain the lacZ gene at theappropriate position, total DNA was digested with Xho I and subjectedSouthern blot hybridization in duplicate. Filters were hybridized withlabeled pkpX2′, which contains wild-type sequences of ICP6 gene. HSV-1wild-type KOS contains a wild-type 2.3 kB Xho I fragment, whereas, hrR3(KOS derived and lacZ insertion mutant in ICP6 gene) contains the 5.3 kBfragment expected if the lacZ gene was inserted. HSV-1 strain F containsan approximately 6.0 kB Xho I fragment due to a polymorphism betweenherpes simplex virus wild-type strains. G207 contains a 9.0 kB fragment,expected if the lac Z gene was inserted into the 6.0 kB fragment ofstrain F. When the filter was hybridized with a lacZ gene probe alone,only the 5.3 kB fragment of hrR3 and the 9.0 kB fragment of G207 wasdetected. These results demonstrates that the lac Z gene fragment isinserted into appropriate site in the genome.

[0101] To confirm that G207 contains deletions in the γ34.5 gene. ViralDNAs of strain F, R3616, G207 were digested with Bam HI and subjected toSouthern blot hybridization. Plasmid pRB4081, containing wild-typesequences of the γ34.5 gene was used as probe. The γ34.5 gene maps inthe Bam HI SP and S fragments. Strain F contains the wild-type Bam HI SPand S versions of these fragments, whereas R3616 and G207 contain thedeleted versions of these fragments. These results demonstrate that bothγ34.5 genes are deleted in R3616 and G207 viral DNA.

[0102] Herpes Simplex Virus Mutants Targeted to Specific Cell Types

[0103] Plasmids containing the 2. 2-kB EGFR promoter fragment frompERCAT2, see Johnson et al., J. Biol. Chem. 263: 5693 (1988), and a2.1-kB BFGF promoter fragment from pF2.ICAT, see Shibata et al., GrowthFactor 4: 277 (1991), are used to characterize transient expression of amarker protein (β-galactosidase). The cell-specificity of theseconstructs is confirmed in human U-87MG glioblastoma cells for BFGF(Takahashi et al., FEBS Letters 288:65 (1991)] and in A431 humanepidermoid carcinoma cells for EGFR. Liberman et al., Nature 313:144(1985). A431 cells are available as ATCC: CRL 1555; U-87 MG MG cells areavailable as ATCC: HTB 14.

[0104] For example, the tumor cell-specific promoter is cloned into anICP4 plasmid upstream of the ICP4 coding region. Examples of ICP4plasmids include pGH108 or pXhoI-C. Roberts et al., J. Virol. 62: 4307(1988). This plasmid is then recombined into herpes simplex virus ICP4⁻.Herpes simplex virus ICP⁻ can be constructed by deletions or insertionsinto the ICP4 coding region. DeLuca et al., J. Virol. 56: 558 (1985);DeLuca and Schaffer, Nucleic Acids Res. 15: 4491 (1987); Paterson andEverett, J. Gen. Virol. 71: 1775 (1990). The vector of the invention canalso be made ICP4- by a deletion or insertion into the ICP4 codingregion. Such ICP4⁻ vectors are isolated on ICP4 expressing cells. DeLucaet al., supra; DeLuca and Schaffer, supra; Paterson and Everett, supra.Alternatively, the ICP4 regulatory region of the herpes simplex virusvector is replaced with the tumor cell-specific promoter so that ICP4 isonly produced in cells capable of expressing the replaced promoter. Theherpes simplex virus mutant containing its ICP4 gene under the controlof a tumor cell-specific promoter is tested for its ability to infectand kill specific tumor cells.

EXAMPLE 2 Safety and Efficacy Studies

[0105] The in vitro efficacy of the mutants as anti-glioma agents can bedetermined using assays of glioma cytotoxicity on cultures of along-term human glioma cell line, U-87MG, as well as early-passage humanglioblastomas. To evaluate tumor inhibition in vivo, subcutaneous U-87MGxenografts in nude mice are treated separately with inoculations of eachviral mutant or vehicle, and tumor growth rates were analyzed. Toinvestigate the potential effects of the herpes simplex virus mutanttreatment on survival, nude mice with intracranial U-87MG xenografts aretreated with virus or vehicle inoculations, and overall survivalis-compared.

[0106] To evaluate the degree of tumor eradication, as well as thepotentially retained neurovirulence of the viruses when used at dosesnecessary to achieve prolonged survival, the brains of long-termsurvivors with intracranial xenografts are sectioned, stained, andmicroscopically examined. For effective in vivo tumor inhibition andsurvival prolongation, careful choice of mutant employing the assaysdescribed herein is essential. The following methods provide clearguidance to those of skill in the art to screen for mutant viral vectorsthat are effective in vivo in inhibiting tumor growth and prolongingsurvival. To establish the relative safety of these viruses as potentialanti-glioma agents, their susceptibility to the common antiherpeticagent ganciclovir is investigated. Finally, to establish the safety ofintracerebral inoculation of the mutant viral vector, animals receive anintracerebral inoculum of the mutant virus and are subsequently assessedfor encephalitis.

[0107] In vitro Cytopathic Killing

[0108] The ability of the herpes simplex virus vectors of the inventionto kill tumor cells is first tested in cell culture. All viral work isdone in approved, designated viral vector rooms. Viruses are initiallygrown on Vero cells, as described in Martuza et al., Science 252:854(1991). To maximize the titer of the viral mutant, the initial viralsuspension was centrifuged at 34,500 g for 2 h at 4° C., and the pelletwas subsequently suspended in media and again titered. Viruses areapplied at varying multiplicities of infection (MOIs), between 10¹ and10⁴. MOI values were calculated from cell number. The appropriate numberof viral pfu was applied and distributed evenly. Coen et al., J. Virol.53: 477 (1985). All viral-infected cell cultures were compared withcontrol cultures (DMEM+ only, no virus). Cells were maintained andobserved microscopically. Cells that had become rounded, losing normalmorphology, and those lifting from the plate were considered dead.Monolayers were considered completely destroyed when 99% or more of thecells exhibited such cytopathic effects.

[0109] Either the mutant or its wild-type parent were applied to a humanglioma line (U-87MG) and African green monkey kidney (Vero) cells atmultiplicities of infection (MOIs) from 10⁻⁴ to 10¹ in DME+ (Dulbecco'smodified Eagle's medium with 1-5% heat-inactivated fetal calf serum((IFCS) and antibiotics). The malignant human glioma line U-87MG wasobtained from American Tissue Cell Collection, Rockville, Md.Additionally, two primary human malignant gliomas were obtained assurgical tumor specimens. Martuza et al., supra, (1991). All cells weregrown in Dulbecco's modified Eagle's medium with 10% fetal bovine serumand antibiotics (DMEM+).

[0110] Subconfluent monolayers of U-87MG and Vero cells were infectedwith the mutant viral vectors of the invention at varying MOIs. Theinfected cells are cultured in 1-5% IFCS-containing medium at 34.5° C.The viable cells were identified by the Trypan blue exclusion method.The mutant expressing cytopathic effects at 24 hours that isproportional to the MOI and expressing >99% cytopathic effect after 10days in U-87MG is deemed to possess the ability to kill glioma cells invitro. The lowest inoculum of the mutant virus that can sustain aspreading infection to destroy the entire monolayer of U-87MG cells willprovide one of the doses at which the mutant is evaluated in vivo. Themutant viral vector also is tested against a different human glioma line(T98G) at various MOIs and assessed for its ability to produce monolayerdestruction within 10 days.

[0111] Short-term glioma cultures were established by explanting threemalignant human gliomas (one anaplastic astrocytoma and twoglioblastomas) obtained at surgery in DME+ and were studied at thesecond passage. The mutants are tested at various MOIs for theircytopathic effects. The herpes simplex virus mutant and dose that iscytopathic in all three primary malignant gliomas is deemed to be ableto kill a wide variety of human brain tumor cells in vitro.

[0112] In addition to glioma cultures, the viral mutants are tested fortheir ability to kill 3 human malignant meningiomas, 1 atypicalmeningioma, 5 neurofibrosarcomas, and 2 medulloblastomas in cellculture, and in the in vivo models. The viral mutants are tested at MOIsranging from 10¹ to 10⁻⁴. Significant tumor inhibition by the mutantvirus reveals a wide range of nervous system tumors for which the viralmutant is efficacious in killing human brain tumor cells.

[0113]FIG. 3 documents the in vitro cytopathic efficacy of G207-1 andG207-2 on U-87MG cells. Subconfluent monolayers of U-87MG cells wereinfected with G207-1 or G207-2 (MOI=0.01 or 1), while the controls weremock-infected and cultured with 10% IFCS-containing medium at 34.5° C.The viable cells were identified by the Trypan blue exclusion method.The number of surviving cells relative to the number of cells inmock-infected control cultures (100%) was assessed. Each data pointrepresents the mean of triplicates. Vertical bars indicate the standarddeviation of the triplicates. Each of the viral mutants killed all ofthe tumor cells by 6 days post-infection. Cytopathic effect appeared onday 1 postinfection for MOI of 1.0, with >99% cytotoxicity evident byday 3 for 1.0 MOI and by day 6 for 0.01 MOI. The cytopathic efficacy ofthese mutants can also be tested on the human glioma cells lines T98G,U-138MG and A172.

[0114] The herpes simplex virus vector of the instant invention can beused to mediate the destruction of other human tumors. Examples of otherhuman tumors that may be amenable to this invention include melanoma,carcinoma of the prostate, breast cancer, pancreatic cancer, lungcancer, colon cancer, lymphoma, hepatoma, and mesothelioma. Human tumorcells can be cultured from primary tumors as described. Fogh and Trempe,HUMAN TUMOR CELLS IN VITRO, Plenum Press, N.Y. (1975) p. 115; Wilson,chapter 8, ANIMAL CELL CULTURE, A PRACTICAL APPROACH. IRL Press (1986).We have shown that a human melanoma cell line, SK-MEL-31 (ATCC: HTB 73);human prostate carcinoma cell lines, Du145 (ATCC: HTB 81) and PC3 (ATCC:CRL 1435); human epidermoid carcinoma cells, A431 (ATCC: CRL 1555); andhuman lung carcinoma cells, Calu-1 (ATCC: HTB54) are susceptible toinfection by attenuated mutants of HSV-1.

[0115] Anti-viral Agent Sensitivity

[0116] To overcome the insensitivity of some of the prior art herpessimplex virus mutants to anti-viral agents, another drug target (forexample, suicide-gene) is inserted into the virus. For example, the CMVUL97 gene (gan^(s); pGMT7-UL97) is inserted into TK HSV-1 mutants andtested for its ability to complement the inability of TK⁻ HSV-1 toreplicate in serum-starved cells and confer ganciclovir sensitivity onthis recombinant. After the viral vector containing the suicide gene istested for ganciclovir sensitivity, a comparison of the ED₅₀ (in vitro)and Mean Survival Time of the suicide containing and suicide absentviral vectors (eg. HSV-1 mutants TK⁻/UL97 and dlsptk) is made in thepresence of ganciclovir.

[0117] Ganciclovir-Sensitivity Assay

[0118] Confluent monolayers of Vero cells in 12-well plates are infectedwith 100 pfu of R3616 or G207, where the MOI remains below 0.0005. Afterremoving the virus inoculum, DMEM plus 1% inactivated fetal calf serumand 1000-fold diluted human immunoglobulin (Armour PharmaceuticalCompany; Kankakee, Ill.) containing various concentrations ofganciclovir is added to triplicate cultures and cells are incubated at37° C. Plaques are visualized by Giemsa stain and counted on day 3postinfection. The ganciclovir (GCV) sensitivity of R3616, G207-1, andG207-2 is illustrated in FIG. 4, which reveals that G207-1 and G207-2are ten times more sensitive to ganciclovir than R3616. The ganciclovirsensitivity of R3616 is similar to wild-type. Each data point representsthe mean of triplicates. The plaque number in the absence of ganciclovirrepresents 100% plaques. The dotted line indicates the ED₅₀ .

[0119] Temperature Sensitivity of Mutant Viral Vector

[0120] To provide an additional safety feature that further compromisesviral replication in the presence of encephalitis and fever, thesensitivity of the mutant viral vectors to temperatures greater than thebasal temperature of the host are ascertained. Table 2 demonstrates thedecreased plaquing efficiency of G207-1 and G207-2 at elevatedtemperatures. The plaque efficiencies were determined by titering virusstocks on Vero cell monolayers. Infected Vero cell monolayers arecultured with 1% IFCS medium at 37° C. or 39.5° C. and fixed at 48 hrpostinfection. Plaques are counted following Giemsa staining. Titers areexpressed as pfu/ml. The hrR3 mutant showed temperature sensitivitycompared to the parental strain KOS as previously reported. Goldsteinand Weller, Virology 166:41 (1988). The HSV-1 wild-type strain F, whichis the parental strain of R3616, G207-1, and G207-2, is also temperaturesensitive. The R3616, G207-1, and G207-2 mutants remain as temperaturesensitive as their parental strains. TABLE 2 Plaquing Efficiencies ofKOS, hrR3, R3616, G207-1, and G207-2 on Vero Cells at 37° C. and 39.5°C. Virus 37° C. 39.5° C. KOS 1.6 × 10⁷ 6.6 × 10⁶ hrR3 3.6 × 10⁸ <10⁴R3616 1.2 × 10⁹ <10⁵ G207-1 6.0 × 10⁷ <10⁴ G207-2 6.0 × 10⁷ <10⁴

EXAMPLE 3 In vito Extracranial Models

[0121] Subcutaneous glioma xenograft Transplantation and Therapy

[0122] The effects of mutant herpes simplex virus infection on humanbrain tumors in vivo were assessed in athymic mice to allow for growthof the human tumors. Subcutaneous xenograft implantation was performedas previously described. Martuza et al., Science 252:854 (1991) andMarkert et. al., Neurosurg. 32:597 (1993). To test the effect of theherpes simplex virus mutants on human glioma in vivo, 1 mm³ mincedglioma pieces (obtained from nude mice previously injectedsubcutaneously with cultured U-87MG cells) are implanted subcutaneouslyinto nude mice. Nude mice are anesthetized with 0.25 ml of a solutionconsisting of 84% bacteriostatic saline, 10% sodium pentobarbitol (1mg/ml), and 6% ethyl alcohol. Animals dying within 48 hours of anyprocedure are considered perioperative deaths and are excluded fromanalysis. Deaths in the subcutaneous tumor experiments are excluded fromanalysis (no significant difference in deaths occurred betweenvirus-treated groups and their corresponding controls).

[0123] Between weeks 4 and 5, animals growing tumors (≧8 mm in diameter)are divided into two groups of 7 to 10 animals per group. Controlsreceived intraneoplastic injections of 50 or 60 μl of DMEM+; treatedanimals received similar intraneoplastic injections of virus suspendedin DMEM+. Doses administered for each virus vary between 10⁶ and 10⁸plaque forming units. Care is taken to distribute virus throughout thetumor. For two-dose experiments, subsequent injections of DMEM+ or virusare made on Day 14. Similar experiments are conducted for each of thevirus mutants at various doses.

[0124] Tumors were measured weekly or twice weekly with Verniercalipers. Growth of subcutaneous xenografts was recorded as the tumorgrowth ratio by formula ([1×w×h]/2)/([1×w×h]_(day0)/2) as described inMartuza et al., supra (1991). Growth ratio comparisons were made at 28days after the initial treatment. Potential differences in growth ratioswere assessed by use of the one-sided Wilcoxon rank test.

[0125] Subcutaneous glioma zenograft Therapy Using G207

[0126] Mice harboring subcutaneous tumors (approximately 6 mm indiameter) were randomly divided (n=6 per group) and treatedintraneoplastically with either 5×10⁷ pfu of G207 virus suspended in0.05ml virus buffer or with buffer alone. The tumor diameter wasmeasured by external caliper measurements. For pathological studies,tumor-bearing mice (>10 mm in diameter) were treated with 1×10⁷ pfu ofG207 and sacrificed on day 8, 15 postinjection. Tumors were removed,placed in fixative for 1 hr and submerged in cold phosphate bufferedsaline. Tumors were then placed overnight in X-gal solution.

[0127]FIG. 5 is a graph showing the growth ratio of subcutaneous U-87MGtumors in Balb/c (nu/nu) mice treated with 5×10⁷ pfu of G207-1 (closedcircle) or G207-2 (closed square) on Day 0, or with control medium alone(open circle). Tumors were measured twice weekly with calipers and thegrowth ratio calculated by dividing the tumor volume by the tumor volumeon the day of initial inoculation. Bars represent mean±SE for eachgroup. The mean tumor growth rate was significantly inhibited in tumorstreated with G207 compared to control tumors treated with medium alone.

[0128] Subrenal Capsule Model

[0129] The effects of G207 on U-87MG cells grown in the subrenal capsuleof the nude mouse also would be tested because the subrenal capsule is asite used for monitoring growth of other nervous system tumors. Lee etal., Neurosurg. 26: 598 (1990); Medhkour et al., J. Neurosurg. 71: 545(1989). U-87MG cells (1.5×10⁶) would be implanted in the subrenalcapsule of nude mice. Ten days later, the tumors are measured andinoculated with varying pfus of G207 in 1 μl DME+ or 1 μl DME+ alone.All mice were re-examined at 14 days and 26 days following inoculationto measure tumor size. Virus-treated tumors that are smaller thancontrol tumors show that the mutant virus is capable of killing tumorcells in vivo.

EXAMPLE 4 In vivo Intracranial Tumor Killing

[0130] To evaluate the in vivo efficacy of the replication-competentherpes simplex virus vector in treating intracerebral gliomas, nude micewould be stereotactically inoculated in the right frontal lobe with1.6×10⁵ U-87MG cells. In a pilot study, a similar cell inoculum caused100% mortality within 1.5 months.

[0131] Ten days after tumor implantation, animals would be dividedrandomly into treatment groups to receive the following therapies at thesame stereotactic coordinates used for the tumor implants: (1) thecontrol group would receive intracranial inoculations of 6 μl DME+ asabove, (2) the second group would receive intracranial inoculations of10³ pfu (low-dose) of the mutant replication-competent viral vector, (3)the third group would receive intracranial inoculations of 10⁵ pfu(middle-dose) of the test virus, and (4) the fourth group would receiveintracranial inoculations of 10⁷ pfu (high dose of the test virus), eachsuspended in 6 μl DME+. Inoculations would be in 2 μl DME+ at thestereotactic co-ordinates initially used to inject the U-87MG cells. By7 weeks, all control animals would be dead, as they have been in pastevaluations. A mutant viral vector of the instant invention is one thatkills intracerebral brain tumors by keeping a significant number of themice alive by seven weeks post-treatment. Significance is determined byplotting experimentals vs. controls in a one-tailed Fischer exact test.

[0132] In vivo Neuropathology

[0133] The animals that remain healthy and neurologically normal at 19weeks are sacrificed. The entire brain will be fixed, serially sectionedat 7 μm intervals, stained with hematoxylin and eosin, andmicroscopically examined for evidence of encephalitis and/or tumor. Theabsence of evidence of encephalitis would reveal that the viral vectorpossesses the characteristic of decreased or attenuated generalizedneurovirulence. The absence of evidence of tumor would reveal that theviral vector is efficacious in killing human brain tumor cells in vivo.

[0134] In vivo treatment would be more effective for those herpessimplex virus mutants that exhibit decreased neurovirulence yet retaincytopathic effects in glioma cells because such vectors would allowtumor treatment at higher viral doses.

[0135] Studies of Herpes Simplex Virus Mutants in Immune CompetentAnimal Models

[0136] To test the efficacy of the herpes simplex virus mutants inkilling human tumor cells in the presence of a competent immune system,the GL261 mouse ependymoblastoma model would be utilized in itssyngeneic host, the C57BL/6 mouse. The GL261 cell line would beimplanted subcutaneously or intracranially in C57BL/6 mice. Animalsharboring subcutaneous GL261 tumors would be randomly divided andtreated intraneoplastically as described above in the nude mouse model.The virus-treated group showing significant growth inhibition, asassessed by the Wilcoxon rank sum test would then be assayed in theintracranial studies.

[0137] For intracranial studies, mice would be injected with 10⁴ GL261cells in the right frontal lobe. After 7 days, the animals would beinoculated intraneoplastically with either mutant virus or with mediumalone. All of the media treated mice would probably die, as they have inprevious studies. The viral mutants that would be capable of prolongingmouse survival to 40 days or longer after tumor cell implantation wouldbe considered efficacious in killing human brain tumor cells in vivo.

[0138] Neuropathology and Tumor Killing in Herpes SimplexVirus-Immunized Animals

[0139] Since herpes simplex virus is endemic in society, an effectivetherapy would have to accommodate patients that have been exposed toHSV-1. Accordingly, it is important to determine whether the mutantherpes simplex virus vectors of the present invention can destroy tumorcells in situ in animals that have been previously immunized to herpessimplex virus. The effect of herpes simplex virus-immunization on theability of the mutant viral vector to kill tumor cells in vivo would betested in the GL261 intracranial model in C57BL/6 mice.

[0140] C57BL/6 mice would be immunized against the KOS strain of herpessimplex virus; another group of mice would be immunized with thewild-type strain from which the vector is derived; another group wouldbe mock-immunized with saline. Those mice that demonstrate high serumtiters of antibody by plaque reduction assay 2 weeks after inoculationwould be used as herpes simplex virus-immunized animals. Four weeksafter immunization, tumor cells would be injected intracerebrally asdescribed above one week later, the tumor would be inoculated at thesame stereotactic coordinates with the vector using medium alone in thenegative control group. The effect of pre-immunization on tumor cellgrowth, subsequent animal death, and the ability of herpes simplex virusto kill the tumor cells would be assessed as described for theintracerebral model.

[0141] In addition, several animals from each group would be sacrificedfor a neuropathological study during each of the acute phase (2 days),subacute phase (1 week), and chronic phase (1 month and 3 months). Thefollowing histologic pathologies would be assessed: tumor size, immunecell infiltration, brain edema, necrosis, alteration of neurons, glia,myelination, hemorrhage, blood vessel proliferation or destruction,reactive astrocytes, normal neurons and glia, ischaemia, gliosis and thespread of virus (PCR for viral DNA or β-galactosidase). These studieswould determine whether pre-immunization against herpes simplex virushas any effect on the mutant viral vector's ability to kill tumor cellsor elicit neuropathogenesis.

[0142] Identification of Virus Location

[0143] Herpes simplex virus containing the E. coli LacZ gene andexpressing β-galactosidase after viral infection is a useful marker forhistologically determining the dynamics and spread of the tagged virus.Because the hrR3 mutant contains the E. coli LacZ gene inserted into theICP6 gene such that the virus expresses B-galactosidase during viralreplication, infected cells can be stained with X-gal. Goldstein andWeller, supra (1988).

[0144] This marker permits following the spread of virus in vivo byexamining brain specimens from mice at various time points afterinfection with hrR3 by staining with X-gal. Kaplitt et al., Mol. & Cell.Neurosci. 2: 320 (1991). The presence of virally-infected cells in fixedbrain sections is determined by PCR and compared to the proportion ofX-gal staining cells. The tumor is visible after counter-staining withH&E or immunohistochemically with tumor-cell or species-specificmarkers. In this way, replication-competent viral vectors would betracked and assessed for their ability to spread to tumor cell depositsat a distance from the main tumor mass. Histologic studies woulddetermine the maximum distance that the virus can spread to reach adistant tumor deposit.

[0145] Another sensitive technique for identifying the presence ofherpes simplex virus or defective herpes simplex virus vector in brainsections would employ PCR. In order to localize viral DNA, DNA for PCRwould be isolated from cells after fixation and histochemistry such thateven single positive cells would generate a specific PCR signal. Usingspecific oligonucleotide primers, unique PCR products would be generatedfrom the viral vector DNA present in these cells. Cover slips would beremoved from slides and small pieces of tissue would be dissected out.The tissue would be incubated with proteinase K, Tween-20,oligonucleotides and PCR buffer at 65° C. for 90 min. and then increasedto 95° C. to inactivate proteinase K. The treated samples would bediluted with dNTPs, PCR buffer and Taq DNA polymerase and thermocycled.The PCR products then would be size analyzed by agarose gelelectrophoresis. In addition, available in situ PCR techniques could beutilized to localize viral DNA during the neuropathological studies.Embretson et al., Proc. Nat'l Acad. Sci. USA 90: 357 (1993).

[0146] Safety of Replication-Competent Herpes Simplex Virus Mutants inMice and Non-human Primates

[0147] To establish that the herpes simplex virus vector does notproduce neurovirulence at the dose required to kill tumor cells, animalsreceive inoculations of tumor-killing doses of the mutant herpes simplexvirus vector to determine whether the vector would cause herpes simplexvirus encephalitis in vivo. Aliquots (10 μl) of G-207-1, G-207-2 andstrain F were inoculated into the right cerebral hemisphere of threeweek old mice; deaths were scored up to 21 days postinfection. Table 3shows that the intracerebral inoculation of Balb/c mice with the parentwild-type virus (strain F) at 10³ p.f.u. caused half the animals to diefrom encephalitis. Chou et al., Science 250: 1262 (1990). The known LD₅₀for strain 17 is also 10³ p.f.u.'s. McKie et al., J. Gen. Virol., 75:733 (1994). In contrast, no mortality or illness was observed followingintracerebral inoculation of the highest titers of G207-1 or G207-2 thatwe could produce (10⁷ p.f.u. in 10 ul). The dose of 10⁷ p.f.u. was shownto kill tumor cells in vivo in the subcutaneous U-87MG tumor growthmodel, as shown in FIG. 5. TABLE 3 Neurovirulence of G207-1 and G207-2in Balb/c mice (i.c. injection for LD₅₀) Balb/c mice (3 wks old)intracranial injection (10 μl) G207-1 1 × 10⁷ pfu/10 μl ×8 (8/8, allmice alive) G207-2 1 × 10⁷ pfu/10 μl ×8 (8/8, all mice alive) Strain F 1× 10³ pfu/10 μl ×8 (4/8, 2 on day 3, 1 on day 5, 1 on day 14)

[0148]Aotus trivigatus, a primate species exceedingly sensitive toherpes simplex virus encephalitis, is used to test the safety of themutant herpes simplex virus vectors of the invention. Katzin et al,Proc. Soc. Exp Biol. Med. 125: 391 (1967); Melendez et al., Lab. Anim.Care 19:38 (1969).

[0149] Magnetic Resonance Imaging (MRI) scanning or other imaginganalysis would be used to assess encephalitis. Monkeys would receive abrain MRI with and without gadolinium prior to the start of the trial.

[0150] Initial testing would be performed at the highest dose that canbe generated for the particular mutant that has been determined to besafe in mice (LD₁₀ or less). For example, 10⁷ pfu would be administeredintracerebrally for the G207 deletion mutant to be tested. The dose thatis well tolerated by a species known to be highly sensitive to herpessimplex virus, provides the most compelling evidence that this treatmentwould be reasonably safe in humans. If no clinical or MRI evidence ofencephalitis is noted within 1 month, another animal would be tested atthat same dose or at a log higher. The animal would be observed dailyfor signs of neurological and systemic illness.

[0151] This method can determine the maximal dose that can safely beadministered intracranially without producing death, persistentneurological signs, or progressive illness. After 12 months, the animalswould be sacrificed and the brains examined for loss or alteration ofneurons, glial reaction, myelination, hemorrhage, blood vesselproliferation or destruction, viral DNA (by PCR) or virally-inducedβ-galactosidase in blood vessels, ischaemia, necrosis, gliosis, andinflammatory reaction. These studies would elucidate the neuropathologiclesions (if any) that might be expected to occur in the normal primatebrain as a result of infection with this vector.

[0152] The genus Aotus had been long thought to be a monotypic genuswith Aotus trivigatus as its sole representative. Studies have proved,however, that Aotus is a multispecific genus with species and subspeciesranging in chromosome number from 2n=46 to 2n=56 (Aotus nancymai,karyotype 1 owl monkey, 2n=54). When the susceptibility of owl monkeysto herpes simplex virus was reported in the 1960's, they could notdistinguish Aotus trivigatus from Aotus nancymai. Malaga et al., Lab.Anim. Sci. 41: 143-45 (1991). Under current taxonomic classification,however, Aotus nancymai was formerly believed to represent Aotustrivigatus. Hershkovitz, Amer. J. Primatol. 4:209 (1983).

[0153] Replication-competent viral vectors of the instant inventionwould be tested for their ability to produce herpes simplex virusencephalitis in primates that are sensitive to herpes simplex virusinduced encephalitis, namely, Aotus nancymai and/or Aotus trivigatus. AnAotus nancymai is still living three weeks after being inoculated with10⁷ pfu of the G207 mutant.

EXAMPLE 5 Treatment of Human Brain Tumors With Replication-CompetentVital Vectors

[0154] Patients with recurrent glioblastoma that was refractory tostandard surgery, radiotherapy and chemotherapy would be treated withherpes simplex virus therapy. The patient would be scanned using MRI orCT or other technique and the tumor and normal brain registered instereotactic space. The virus would be administered usingstereotactically guided neurosurgical techniques. A computer tomography(CT) scan or magnetic resonance imaging (MRI) scan computes thestereotactic frame that would be used to accurately inoculate virus intoa tumor at one or more locations. Virus would be inoculated at a dose of10¹ to 10⁷ p.f.u. per inoculation using a <2 mm cannula. The number ofsites inoculated would depend on the size of the tumor. Patients wouldbe followed with periodic MRI scans and with neurological examination,blood count, and liver function tests.

[0155] In an alternate scheme, patients will be operated to remove muchof the recurrent tumor and virus will inoculated in the resected tumorbed in a fashion similar to above.

EXAMPLE 6 Replication-Competent Herpes Sixplex Virus Vector Vaccines

[0156] The herpes simplex virus vector of the invention can be used as avaccine to protect an animal against herpes simplex virus infection. Inthe present context, “protecting” a subject against herpes simplex virusincludes both (1) a prophylactic vaccine, i.e., a vaccine used toprevent a future herpes simplex virus infection, and (2) a therapeuticvaccine for treating an existing herpes simplex viral infection.

[0157] The herpes simplex virus sample would be prepared using standardmethodology. Herpes simplex virus-infected Vero cells would be frozen at−70° C. until they are to be used. The material would be thawed and thecell debris would be pelleted by centrifugation. The supernatant fluidwould be discarded and the pellet resuspended to its original volume.This material would most closely approximate that used in vaccinemanufacture. This suspension would be sonicated twice for 20 seconds.

[0158] Herpes simplex virus plaque titers would be determined bystandard procedures. For example, the virus would be titrated intriplicate on monolayers of Vero cells in 6-well plates. Afteradsorption of samples for 2 hours, cells would be overlayed with mediacontaining 0.6% agarose and incubated at 37° C. in a CO₂-richenvironment for 48 h. A second overlay, the same as above except foraddition of neutral red, would be added and the cells would be incubatedan additional 24 hours.

[0159] The herpes simplex virus pools would be titrated beforefiltration. The pools then would be filtered through a Nalgene 0.45 μmfilter, sampled, refiltered through a second filter and then resampled.

EXAMPLE 7 Testing of Herpes Simplex Virus-Vaccine for Pathogenicity in aMurine Model and Monkey Model

[0160] The lethality of the herpes simplex virus vaccine would becompared with the lethality of other herpes simplex virus vaccines in<24 h old suckling mice, CD-1 strain, (Charles River, Raliegh, N.C.).Meignier et al., J. Infect. Diseases 158: 602 (1988); Burke, Curr.Topics in Microbiology and Immunology 179: 137 (1992). Comparativetitration of herpes simplex virus vector vaccine and wild type vaccineswould be conducted in a single test using the final bulk of the herpessimplex virus vaccine.

[0161] Logarithmic dilutions of the vaccine would be prepared. Twolitters of 5 mice each would be used for each dilution. Mice would beinoculated intracerebrally with 0.03 ml of the appropriate dilutions andobserved for 21 days. Mouse lethality would be calculated as the dose inpfu that killed 50% of mice (e.g., pfu/0.03 ml of vaccine divided byLD₅₀ of vaccine).

[0162] Also, the herpes simplex virus vector would be given to 4 monkeysin the study. An additional six monkeys would receive the vector oneyear after immunization with the herpes simplex virus vector of theinvention. If intradermal and subcutaneous administration of the vaccinecandidate is well tolerated, the herpes simplex virus vector vaccine isdeemed to be safe for use as an immunoprotective agent against herpessimplex virus.

[0163] In addition, all monkeys would be tested for serum antibodytiters specific for herpes simplex virus. Monkey seroconversion would bemeasured by ELISA, after primary immunization. If all monkeysseroconvert, the herpes simplex virus vector vaccine is deemed to haveefficacy as an immunoprotective agent against herpes simplex virus.

EXAMPLE 8 Human Clinical Studies with Herpes Simplex Virus VectorVaccine

[0164] For use as a vaccine, the mutated herpes simplex virus vector ofthe invention would be inoculated subcutaneously. Thereafter, herpessimplex virus-specific antibody titers and herpes simplex virus-specificcell mediated response levels would be determined. Meignier et al., J.Infect. Diseases 162: 313 (1990); Burke, Curr. Topics in Microbiologyand Immunology 179: 137 (1992). The preliminary phase of the study wouldinvolve an inoculation of four individuals with documented HSV-1infections (Group 1), succeeded by inoculation of four HSV-1-naiveindividuals (Group 2) 21 days after the first group had been inoculated.Previous HSV-1 exposure would be documented by medical records orunequivocal HSV-1 outbreak, as assessed by HSV-1 immunofluorescenceassay available in clinical laboratories. This would be followed by arandomized trial in 24 herpes simplex virus-naive volunteers (Group 3).Anti HSV-1 immune globulin and anti-herpetic agents are available onsite for the treatment of serious adverse reactions.

[0165] Group 1, 2, 3 and 4 subjects would be admitted to the hospitalthree days prior to inoculation and would remain as inpatients untilfour days after inoculation. Subjects would then be discharged andassessed on an outpatient basis with clinical examinations for potentialreactions or complications through day 21. Subjects developing fever,rash, lethargy, necrotic skin lesions, or neurologic signs are followedwith subsequent daily clinical examinations and admitted to the hospitalif deemed necessary.

[0166] Group 5 volunteers, all HSV-1-naive, would be enrolled dependingon availability as outpatient subjects. Group 5 volunteers would berandomly assigned to one of two subgroups: one would receive a singleinjection and the other would receive a booster.

[0167] Protocol participation studies would include periodicexaminations of the following: CBC with differential and platelets,urinalysis, serum chemistries, serum viremia, serum herpes simplex virusantibody, and lymphocyte immune responses to herpes simplex virusantigen. Remaining serum samples would be maintained frozen at −80° to−120° C. and available for additional studies and/or repeats of selectedstudies as needed. Fluid in vesicular or weeping lesions at the site ofinoculation or at distant sites would be sampled and placed in viralisolation transport medium to attempt virus recovery. Serum antibodydeterminations would include ELISA reactivity with cells infected withthe herpes simplex virus vector, HSV-1 antigen and plaque reductionneutralization of HSV-1 viral vector.

[0168] Clinical trials of the herpes simplex virus vector should showthe vaccine to be safe and effective in humans. Vaccine recipients wouldbe expected to produce significant humoral response as measured byELISA. A positive response would be characterized by the production ofboth neutralizing and non-neutralizing antibodies, the latter beingmeasured by plaque reduction and neutralization assays. In addition,positive lymphocyte blastogenesis assays would be expected todemonstrate that lymphocytes from vaccine recipients proliferate andproduce cytokines upon exposure to herpes simplex virus antigen invitro.

1-15. (Canceled)
 16. A herpes simplex virus with a genome that comprises(i) an expressible non-herpes simplex virus nucleotide sequence encodinga desired protein and (ii) an alteration, relative to wild type, in theγ34.5 gene.
 17. The herpes simplex virus of claim 16, wherein bothcopies of said γ34.5 gene are altered, relative to wild type.
 18. Theherpes simplex virus of claim 16, further comprising at least onefurther gene alteration, relative to wild type.
 19. The herpes simplexvirus of claim 18, wherein said at least one further gene alteration isin the ribonucleotide reductase gene.
 20. The herpes simplex virus ofclaim 16, wherein said herpes simplex virus is G207.
 21. The herpessimplex virus of claim 16, wherein said protein is a cytokine.
 22. Theherpes simplex virus of claim 16, wherein said virus is targeted to atumor cell of non-nervous tissue origin.
 23. The herpes simplex virus ofclaim 22, wherein said tumor cell is a neural tumor cell.
 24. The herpessimplex virus of claim 16, wherein said virus is targeted to a specifictumor type with a tumor cell-specific promoter.
 25. The herpes simplexvirus of claim 24, wherein said promoter is nestin promoter.
 26. Theherpes simplex virus of claim 24, wherein said promoter is basicfibroblast growth factor promoter.
 27. The herpes simplex virus of claim24, wherein said promoter is epidermal growth factor promoter.
 28. Theherpes simplex virus of claim 16, wherein an essential viral geneproduct of said virus is under the control of a tumor cell-specificpromoter rather than its own viral promoter.
 29. A compositioncomprising the herpes simplex virus of claim 16 and a pharmaceuticallyacceptable vehicle for said virus.